Comprehensive quantitative modeling of translation efficiency in a genome‐reduced bacterium

Abstract Translation efficiency has been mainly studied by ribosome profiling, which only provides an incomplete picture of translation kinetics. Here, we integrated the absolute quantifications of tRNAs, mRNAs, RNA half‐lives, proteins, and protein half‐lives with ribosome densities and derived the initiation and elongation rates for 475 genes (67% of all genes), 73 with high precision, in the bacterium Mycoplasma pneumoniae (Mpn). We found that, although the initiation rate varied over 160‐fold among genes, most of the known factors had little impact on translation efficiency. Local codon elongation rates could not be fully explained by the adaptation to tRNA abundances, which varied over 100‐fold among tRNA isoacceptors. We provide a comprehensive quantitative view of translation efficiency, which suggests the existence of unidentified mechanisms of translational regulation in Mpn.


Appendix Figure S2. Reproducibility of ribosome counts and ribosome density and their dependence on mRNA levels.
(A) Ribosome counts per gene for the two standard growth replicates rep1 and rep2, and pearson correlation coefficient (inset).
(B) Distribution of average ribosome density in CDSs for the replicate rep1, for CDSs stratified into four groups of normalized RNA expression level, from lowest (left) to highest (right).Ribosome density was overall not related to the level of mRNA expression, as genes with low mRNA levels had a very similar distribution of ribosome density compared to genes with high mRNA levels (C) The reproducibility of ribosome density at gene level depends on mRNA levels.Correlation of average ribosome density in CDSs for the two replicates rep1 and rep2, for CDSs stratified into four groups of normalized RNA expression level, from lowest (left, top) to highest (right, bottom).Pearson correlation coefficient in linear space and nb of CDSs are displayed for each group.Because mRNA level is a denominator in the expression of the ribosome density, genes with low mRNA levels are particularly sensitive to noise and displayed a lower correlation between replicates (r = 0.81) compared to genes with higher mRNA levels (r = 0.95).
(B) Absolute translation efficiency for genes classified by the presence of a ribosome binding site (RBS).The strength of the affinity of the RBS sequence to the anti-Shine-Dalgarno motif CCUCCU was determined by computing the hybridization energy and classified into 3 bins (low, medium and strong affinity).Precision of the translation efficiency was higher for proteins which were quantified by labeled peptides (labeled), compared to proteins quantified by label-free mass spectrometry (not labeled).Only genes which are at the first position in their operon were selected.

Appendix Figure S6. Correlation between the absolute translation efficiency and the amino acid identity at the N-terminal.
(C) Translation efficiency to ribosome density ratio and folding energy along the CDS.The folding energy was computed along the CDS in a rolling window of size 60 nt and averaged over the CDS, and then categorized into 3 bins: low (< -11.6 kcal/mol), medium (< -7.82 kcal/mol) and high (> -7.82 kcal/mol).
(C) Correlation between the absolute translation efficiency and the tRNA adaptation index (tAI) of the first 30 codons at the N-terminal.(D) Absolute translation efficiency of genes encoding for ribosomal proteins compared to other genes.(E) Correlation between the absolute translation efficiency and the length of the 5'UTR.Only genes which are at the first position on their operon were selected.Appendix Figure S4.Correlation between the absolute translation efficiency and other features of the mRNA.(A) Absolute translation efficiency of genes classified by leaderless-like/leadered.(B) Correlation between the absolute translation efficiency and the nucleotide composition of the 5'UTR close to the start codon.Only genes which are at the first position in their operon were selected.The A content was computed in a window of 15 nt upstream the start codon.A small jitter was applied to the x position of data points for easier visualization.(C) Correlation between the absolute translation efficiency and the RNA degradation rate.Appendix Figure S5.Correlation between the absolute translation efficiency and the nucleotide identity in the 5'UTR sequence at specific positions upstream of the start codon.
(D-E) Correlation between translation efficiency to ribosome density ratio and internal SD-like motifs.The possible influence of internal SD-like motifs was tested in two ways.(E) The count of internal SD-like motifs within the CDS.(E) The sum of the free energies of each SD-like motif within the CDS.(F) Correlation between the translation efficiency to ribosome density ratio and the RNA degradation rate.Appendix Figure S14.Correlation between the translation efficiency to ribosome density ratio and the GC content of the CDS.(A) GC content of the CDS (B) GC content at the first nucleotide position of codons (GC1) (C) GC content at the second nucleotide position of codons (GC2) (D) GC content at the third nucleotide position of codons (GC3).